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Mitotic phosphorylation activates hepatoma-derived growth factor as a mitogen
Allen D Everett1, Jun Yang, Monzur Rahman
1Department of Pediatrics, Cardiology Division, Johns Hopkins University, 600 N. Wolfe Street, Baltimore, MD 21287, USA. aeveret3@jhmi.edu
BMC Cell Biology
|April 15, 2011
Summary
Hepatoma-derived growth factor (HDGF) is phosphorylated at S103 during mitosis, a modification crucial for its mitogenic function in vascular smooth muscle cells. This phosphorylation is vital for cell cycle progression.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Hepatoma-derived growth factor (HDGF) is a known mitogen for various cell types.
- Previous studies identified HDGF as a phosphoprotein using mass spectrometry but lacked functional validation.
- The functional significance of HDGF post-translational modifications remained unclear.
Purpose of the Study:
- To investigate the phosphorylation of HDGF in vascular smooth muscle cells (VSMC).
- To determine the functional consequence of HDGF phosphorylation, particularly at specific serine residues.
- To elucidate the role of HDGF phosphorylation in cell cycle regulation and proliferation.
Main Methods:
- Primary mouse aortic VSMC were used to study HDGF phosphorylation.
- Site-directed mutagenesis was employed to substitute serine residues (S103, S165, S202) with alanine.
- A phospho-specific antibody against S103-phosphorylated HDGF was developed.
- Flow cytometry (FACS) analysis was performed to assess cell cycle progression.
- Cells were treated with nocodazole to induce mitotic arrest.
Main Results:
- HDGF was confirmed to be phosphorylated in primary mouse aortic VSMC.
- Substitution of S103, S165, and S202 with alanine reduced HDGF phosphorylation.
- Mitosis-specific phosphorylation of HDGF at S103 was demonstrated using a specific antibody.
- HDGF-S103A mutants exhibited impaired mitogenic activity and caused G2/M cell cycle arrest.
- Cells expressing HDGF-S103D (an phosphomimetic mutant) showed normal cell cycle progression.
- Nocodazole treatment significantly increased S103 phosphorylation in VSMC.
Conclusions:
- HDGF is a phosphoprotein, with S103 phosphorylation being mitosis-related.
- Phosphorylation of S103 is essential for HDGF's mitogenic function.
- Cell cycle-regulated phosphorylation of HDGF likely plays a significant role in vascular cell proliferation.
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